LIF-JAK1-STAT3信号通路延迟人角膜内皮细胞接触抑制作用机制研究

发布时间：2018-02-11 18:30

本文关键词： 角膜内皮细胞 LIF STAT3 接触抑制增殖 E2F2 p16~(INK4a)　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：本研究旨在探究人角膜内皮细胞在改良胚胎干细胞培养基体外培养中能持续增殖并延迟接触抑制的相关机制。方法：通过比较人角膜内皮单层细胞在SHEM, ESCM, ESCM+LIF, ESCM+bFGF和MESCM等不同培养基中培养第7,21,35和42天后5-溴脱氧尿嘧啶核苷染色情况来判断人角膜内皮细胞在不同环境下的增殖能力。并通过qRT-PCR检测和定量分析胚胎干细胞标志物,神经嵴细胞标志物,骨形成蛋白配体和受体以及细胞周期相关基因的表达情况。通过共聚焦荧光染色显微镜等技术观察相关信号通路关键蛋白在细胞不同部位的表达情况,从而明确人角膜内皮细胞延迟接触抑制的相关信号通路机制。结果：人角膜内皮细胞在SHEM培养基中第21天时5-溴脱氧尿嘧啶核苷染色阴性,而在ESCM+LIF和MESCM中第35天时仍染色阳性。另外,在ESCM中添加白血病抑制因子(LIF, leukemia inhibitory factor)不仅和MESCM一样可以明显增加其中7种胚胎干细胞和神经嵴细胞标志物的表达,还可以明显增加Oct4, Rexl和SSEA4等胚胎干细胞标志物的表达。在ESCM中单独或同时添加LIF和bFGF都不足以激活经典或非经典的BMP信号通路。进一步分析发现磷酸化的STAT3核转位只有在包含LIF的培养基中才存在,提示LIF激活了培养的人角膜内皮细胞中的LIF-JAK1-STAT3信号通路。第35天时通过JAK1和STAT3siRNA的基因敲减技术处理可以消除人角膜内皮细胞在LIF包含的培养基中阳性的5-溴脱氧尿嘧啶核苷染色和G1/S细胞周期相关基因p-Rb等的核转位,进一步证实延迟人角膜内皮细胞的接触抑制确实受LIF-JAK1-STAT3信号通路的调控。结论：和SHEM相比,MESCM培养基可以通过延迟人角膜内皮细胞接触抑制的重建促进角膜内皮细胞的体外增殖生长。其中LIF而不是bFGF可以激活LIF-JAK1-STAT3信号通路并通过促进G1/s细胞周期正向基因的表达,从而达到延迟人角膜内皮细胞接触抑制的作用。
[Abstract]:Aim: to investigate the mechanism of human corneal endothelial cells proliferation and delayed contact inhibition in vitro. Methods: human corneal endothelial monolayer cells were cultured in different culture medium, such as Shem, ESCM, ESCM LIF, ESCM bFGF and MESCM. The 5-bromodeoxyuridine (5-bromodeoxyuridine) staining of human corneal endothelial cells in different environment was determined by comparing the 5-bromodeoxyuridine (5-bromodeoxyuridine) staining of human corneal endothelial monolayer cells cultured in different media. And to detect and quantify the markers of embryonic stem cells by qRT-PCR, The expression of neural crest cell markers, bone morphogenetic protein ligands and receptors, and cell cycle related genes were observed by confocal fluorescent staining. Therefore, the mechanism of delayed contact inhibition in human corneal endothelial cells was clarified. Results: human corneal endothelial cells were negative in 5-bromodeoxyuridine staining on the 21st day in SHEM medium, but still positive in ESCM LIF and MESCM on the 35th day. The addition of Leukemia Inhibitors (LIF, leukemia inhibitory Factor) to ESCM not only increased the expression of seven markers of embryonic stem cells and neural crest cells, but also increased the expression of LIF, leukemia inhibitory factor and MESCM. The expression of ESC markers such as Oct4, Rexl and SSEA4 were significantly increased. The addition of LIF and bFGF in ESCM alone or at the same time was not sufficient to activate the classical or non-classical BMP signaling pathway. Further analysis revealed that phosphorylated STAT3 nuclear transduction was not sufficient. The site only exists in the medium containing LIF, The results suggest that LIF activates the LIF-JAK1-STAT3 signaling pathway in cultured human corneal endothelial cells. At day 35, the positive 5-bromodeoxyuridine of human corneal endothelial cells in LIF medium can be eliminated by JAK1 and STAT3siRNA gene knockout technique. Nucleoside staining of pyrimidine and nuclear translocation of p-Rb and other genes associated with cell cycle of G1 / S cells. It is further confirmed that the delayed contact inhibition of human corneal endothelial cells is indeed regulated by the LIF-JAK1-STAT3 signaling pathway. Conclusion: compared with SHEM, MESCM can promote the proliferation and growth of corneal endothelial cells by delaying the reconstitution of human corneal endothelial cell contact inhibition in vitro. LIF, not bFGF, can activate the LIF-JAK1-STAT3 signaling pathway and promote G1 / s. Cell cycle forward gene expression, In order to delay the contact inhibition of human corneal endothelial cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R772.2

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